Anatomy of a simple acyl intermediate in enzyme catalysis: combined biophysical and modeling studies on ornithine acetyl transferase.
Acyl-enzyme complexes are intermediates in reactions catalyzed by many hydrolases and related enzymes which employ nucleophilic catalysis. However, most of the reported structural data on acyl-enzyme complexes has been acquired under noncatalytic conditions. Recent IR analyses have indicated that so...
| Main Authors: | , , , , , , , |
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| Format: | Journal article |
| Language: | English |
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2009
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| _version_ | 1826299815795884032 |
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| author | Iqbal, A Clifton, I Bagonis, M Kershaw, N Domene, C Claridge, T Wharton, C Schofield, C |
| author_facet | Iqbal, A Clifton, I Bagonis, M Kershaw, N Domene, C Claridge, T Wharton, C Schofield, C |
| author_sort | Iqbal, A |
| collection | OXFORD |
| description | Acyl-enzyme complexes are intermediates in reactions catalyzed by many hydrolases and related enzymes which employ nucleophilic catalysis. However, most of the reported structural data on acyl-enzyme complexes has been acquired under noncatalytic conditions. Recent IR analyses have indicated that some acyl-enzyme complexes may be more flexible than most crystallographic analyses have implied. OAT2 is a member of the N-terminal nucleophile (Ntn) hydrolase enzyme superfamily and catalyzes the reversible transfer of an acetyl group between the alpha-amino groups of ornithine and glutamate in a mechanism proposed to involve an acyl-enzyme complex. We have carried out biophysical analyses on ornithine acetyl transferase (OAT2), both in solution and in the crystalline state. Mass spectrometric studies identified Thr-181 as the residue acetylated during OAT2 catalysis; (13)C NMR analyses implied the presence of an acyl-enzyme complex in solution. Crystallization of OAT2 in the presence of N-alpha-acetyl-L-glutamate led to a structure in which Thr-181 was acetylated; the carbonyl oxygen of the acyl-enzyme complex was located in an oxyanion hole and positioned to hydrogen bond with the backbone amide NH of Gly-112 and the alcohol of Thr-111. While the crystallographic analyses revealed only one structure, IR spectroscopy demonstrated the presence of two distinct acyl-enzyme complex structures with carbonyl stretching frequencies at 1691 and 1701 cm(-1). Modeling studies implied two possible acyl-enzyme complex structures, one of which correlates with that observed in the crystal structure and with the 1691 cm(-1) IR absorption. The second acyl-enzyme complex structure, which has only a single oxyanion hole hydrogen bond, is proposed to give rise to the 1701 cm(-1) IR absorption. The two acyl-enzyme complex structures can interconvert by movement of the Thr-111 side-chain alcohol hydrogen away from the oxyanion hole to hydrogen bond with the backbone carbonyl of the acylated residue, Thr-181. Overall, the results reveal that acyl-enzyme complex structures may be more dynamic than previously thought and support the use of a comprehensive biophysical and modeling approach in studying such intermediates. |
| first_indexed | 2024-03-07T05:07:41Z |
| format | Journal article |
| id | oxford-uuid:da7c7288-b39e-40b8-b7bc-f6ff73cf06fb |
| institution | University of Oxford |
| language | English |
| last_indexed | 2024-03-07T05:07:41Z |
| publishDate | 2009 |
| record_format | dspace |
| spelling | oxford-uuid:da7c7288-b39e-40b8-b7bc-f6ff73cf06fb2022-03-27T09:03:34ZAnatomy of a simple acyl intermediate in enzyme catalysis: combined biophysical and modeling studies on ornithine acetyl transferase.Journal articlehttp://purl.org/coar/resource_type/c_dcae04bcuuid:da7c7288-b39e-40b8-b7bc-f6ff73cf06fbEnglishSymplectic Elements at Oxford2009Iqbal, AClifton, IBagonis, MKershaw, NDomene, CClaridge, TWharton, CSchofield, CAcyl-enzyme complexes are intermediates in reactions catalyzed by many hydrolases and related enzymes which employ nucleophilic catalysis. However, most of the reported structural data on acyl-enzyme complexes has been acquired under noncatalytic conditions. Recent IR analyses have indicated that some acyl-enzyme complexes may be more flexible than most crystallographic analyses have implied. OAT2 is a member of the N-terminal nucleophile (Ntn) hydrolase enzyme superfamily and catalyzes the reversible transfer of an acetyl group between the alpha-amino groups of ornithine and glutamate in a mechanism proposed to involve an acyl-enzyme complex. We have carried out biophysical analyses on ornithine acetyl transferase (OAT2), both in solution and in the crystalline state. Mass spectrometric studies identified Thr-181 as the residue acetylated during OAT2 catalysis; (13)C NMR analyses implied the presence of an acyl-enzyme complex in solution. Crystallization of OAT2 in the presence of N-alpha-acetyl-L-glutamate led to a structure in which Thr-181 was acetylated; the carbonyl oxygen of the acyl-enzyme complex was located in an oxyanion hole and positioned to hydrogen bond with the backbone amide NH of Gly-112 and the alcohol of Thr-111. While the crystallographic analyses revealed only one structure, IR spectroscopy demonstrated the presence of two distinct acyl-enzyme complex structures with carbonyl stretching frequencies at 1691 and 1701 cm(-1). Modeling studies implied two possible acyl-enzyme complex structures, one of which correlates with that observed in the crystal structure and with the 1691 cm(-1) IR absorption. The second acyl-enzyme complex structure, which has only a single oxyanion hole hydrogen bond, is proposed to give rise to the 1701 cm(-1) IR absorption. The two acyl-enzyme complex structures can interconvert by movement of the Thr-111 side-chain alcohol hydrogen away from the oxyanion hole to hydrogen bond with the backbone carbonyl of the acylated residue, Thr-181. Overall, the results reveal that acyl-enzyme complex structures may be more dynamic than previously thought and support the use of a comprehensive biophysical and modeling approach in studying such intermediates. |
| spellingShingle | Iqbal, A Clifton, I Bagonis, M Kershaw, N Domene, C Claridge, T Wharton, C Schofield, C Anatomy of a simple acyl intermediate in enzyme catalysis: combined biophysical and modeling studies on ornithine acetyl transferase. |
| title | Anatomy of a simple acyl intermediate in enzyme catalysis: combined biophysical and modeling studies on ornithine acetyl transferase. |
| title_full | Anatomy of a simple acyl intermediate in enzyme catalysis: combined biophysical and modeling studies on ornithine acetyl transferase. |
| title_fullStr | Anatomy of a simple acyl intermediate in enzyme catalysis: combined biophysical and modeling studies on ornithine acetyl transferase. |
| title_full_unstemmed | Anatomy of a simple acyl intermediate in enzyme catalysis: combined biophysical and modeling studies on ornithine acetyl transferase. |
| title_short | Anatomy of a simple acyl intermediate in enzyme catalysis: combined biophysical and modeling studies on ornithine acetyl transferase. |
| title_sort | anatomy of a simple acyl intermediate in enzyme catalysis combined biophysical and modeling studies on ornithine acetyl transferase |
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